SAHLPA-23 was more highly correlated with all study variables than SAHLPA-18. Although both instruments displayed acceptable discriminative power, SAHLPA-23 had better accuracy than SAHLPA-18 . The SAHLPA-23 is an independent, feasible and innovative tool for estimation of health literacy in the Portuguese adult population.The goal of this study was to adapt, improve and validate a short, self-administered health literacy assessment tool for European Portuguese-speaking adults. Health literacy tools are of great importance to health authorities and professionals, as low or inadequate health literacy, that is, a limited capacity to handle health-related information, is associated with higher morbidity and mortality. The 18-item Short Assessment of Health Literacy for Brazilian Portuguese-speaking adults (SAHLPA-18) was adapted into European Portuguese. The European Portuguese tool (SAHLPA-23) includes five additional items. The SAHLPA-23 was tested in a convenience sample of 503 participants from two Portuguese regions. Socio-demographic data, literacy and cognitive indicators were collected. Participants also completed a questionnaire on comprehension of written health materials. Construct validity was assessed through correlations between SAHLPA-23 scores and education, literacy, and cognitive variables and score on the comprehension questionnaire. The psychometric properties of the new tool were compared with those of the SAHLPA-18. The mean SAHLPA-18 and SAHLPA-23 scores were 13.9  and 18.3 , respectively. Both tools showed adequate reliability (Cronbach’s  In the late 20th century literacy was mainly understood in terms of reading and writing skills, but the concept has been extended and now includes the ability to interpret texts, perform calculations and participate actively in society : participants are required to read and recognise health words .The Test of Functional Health Literacy in Adults : participants are required to perform a modified cloze procedure  .The Newest Vital Sign : participants are required to answer comprehension questions about the nutrition label for an ice cream .The Short Assessment of Health Literacy - Spanish and English versions : participants are required to read and recognise health words .Various tools and methodological approaches have been used to evaluate health literacy  ; this demonstrates that the decision-makers have recognised the population’s low level of health literacy and the need to improve it. In addition, Portuguese, taking the European, Brazilian, African and Asian varieties together, is the world’ seventh most commonly spoken language (with around 200 million of speakers) , ‘adverse reactions’ (efeitos secundários), ‘excipient’ (excipiente), ‘fertility’ (fertilidade) and ‘precautions’ (precauções). Two of the five new items were deliberately designed with two-word targets in European Portuguese (‘active ingredient’ and ‘adverse reactions’) as it was thought that longer targets would provide better discrimination between levels of health literacy. The positioning of the five additional items in the tool is random  and assess comprehension of health-related documents. Comprehension was evaluated with two questionnaires on the comprehension of PLs of medicinal products. These last two questionnaires were developed in accordance with the European guidelines on medicine PLs clarity and comprised eight questions  Europea. They wecarro (car), vaso (vase), bola , lápis (pencil) and relógio (watch) (maximum score: 5 points).The spelling of the words et al., The mental calculus task: 100−7(maximum score=1 point), plus two additional calculation tasks (maximum score=2 points). The two additional calculation tasks were as follows: a sum followed by a subtraction to calculate how much money respondents will get back in a purchase and a division to calculate the price of a certain amount of a product. These last two calculation tasks were adapted from the last national Portuguese literacy study  books ; and (2) magazines or journals . Participants’ writing habits were also assessed, using questions about how often they performed simple writing tasks . The response options were as follows: daily, at least once a week, at least once a month, rarely and never (maximum score: 4 points). These questions were also adapted from the last national Portuguese literacy study 1995, reported by Benavente et al. . The maxet al., Before administering the SAHLPA-23 and the study questionnaires a brief screening test was administered to detect any cognitive problems  that might affect participants’ judgement and reasoning capacity. The test consisted of the following questions and simple tasks: (1) ‘What month is it?’ (2) ‘Where are we ?’ (3) ‘The researcher is about to say three words in loud voice: please write them down ,’ (4) ‘How much is 100 minus 7?’ (5) The researcher shows the participant a pencil and asks him or her to write down the name of the object, (6) The researcher shows the participant a watch and asks him or her to write the name of the object and (7) ‘Write down the three words that the researcher said in loud voice a few minutes ago, again.’ These questions and tasks were based on the full version of the Mini-Mental State Examination  The discriminatory ability of both tests was examined using receiver operating characteristic (ROC) curves and area under the curve (AUC). The optimal cut-off point was defined as the maximum value of the Youden index, calculated as: J=max [sensitivity c+specificity c–1], where c represents all possible criterion values  exploratory factor analysis (EFA) was carried out [Kaiser–Meyer–Olkin Measure (KMO) of sampling and adequacy and the Bartlett’s Test of Sphericity]. Principal component factor analysis with oblique rotation  was conducted based on eigenvalues values  .A cut-off score for adequate health literacy was identified using ROCs for the established literacy tasks. This procedure resulted in exclusion of some items and the definition of a cut-off score for distinguishing between adequate and inadequate health literacy. (4) Spearman’s rank test was used to assess correlations between score on the new short health literacy tool (SAHLPA-23) and the construct variables. Construct validity was evaluated by calculating correlations between SAHLPA-23 scores and the following variables: years of schooling, cognitive index, literacy tasks, reading and writing habits, score on the comprehension questionnaire, and the socio-demographic variables. The Statistical Package for Social Sciences (SPSS) version 22  was used for all analyses except for ROC analyses, which were conducted using MedCalc version 14.8 . All statistical tests were two-tailed  were male, with a mean age of 38.7 year  and an average of 10.3 years (SD=4.8) of schooling , the military (21.3%) and university undergraduates  (19.9%). The remaining participants were evenly distributed across the other institutions. Overall, 49.3% of the participants were from Lisbon and Tagus Valley, 53.1% (chooling . Table 3χ2(253)=2764.4, P<0.001], indicating that the correlation matrix was suitable for factor analysis. According to the ratio-of-first-to-second-eigenvalues-greater-than-three rule  the SAHLPA-23 displayed a one-dimensional structure (5.97/1.78=3.36).The SAHLPA-23 data yielded a good KMO score (0.89) and Bartlett’s sphericity test was significant , indicating that the correlation matrix was suitable for factor analysis. As with the SAHLPA-23, the ratio-of-first-to-second-eigenvalues-greater-than-three rule also indicated that a one-dimensional structure (4.97/1.52=3.32). Visual inspection of the scree plots for both instruments also revealed a multidimensional factor structure  and Bartlett’s sphericity test was significant , the cognitive index [rs(482)=0.374], the comprehension questionnaire [rs(482)=0.561], direct measures of literacy [rs(482)=0.308] and indirect measures of literacy [rs(482)=0.234], suggesting that the SAHLPA-23 had good construct validity. The SAHLPA-23 was more highly correlated with all relevant study variables, except the direct and indirect measures of literacy, than the SAHLPA-18 . Similarly, the association with the direct literacy index was larger in the case of the SAHLPA-23 than the SAHLPA-18 . The rest of the variables were similarly associated with both instruments (P>0.05).Fisher In accordance with classical data reduction procedures , which suggests the tool is a moderately accurate method of detecting inadequate health literacy. The recommended minimum AUC for diagnostic tests is 0.75  (maximum score=24) on our structured protocol for comprehension of PLs were defined has having inadequate health literacy. This cut-off score was based on the recommendations of the European Medicines Agency as expressed in the  Larner, .Figure 1J=0.42; sensitivity=68.2%; specificity=73.5%)  . This cu et al., .Z=3.36, P<0.001. The SAHLPA-23 also had a larger AUC than the direct measure of literacy, ΔAUC=0.10, Z=3.40, P<0.001 and the indirect measure of literacy, ΔAUC=0.15, Z=4.70, P<0.001 . Comparisons of the ROC curves for SAHLPA-23 and other instruments are shown in Years of formal schooling  had the highest AUC, which was higher than the AUC for the SAHLPA-23  were classed as having inadequate health literacy with the SAHLPA-23. This figure is slightly higher than that obtained in the last Portuguese health literacy study, in which only 38.5% of the 1001 participants were classified has having adequate or excellent health literacy  was higher than the mean score on the direct literacy index (numeracy tasks), it is likely some participants overestimated their reading and writing habits . Additionally, writing habits were evaluated based on self-reported frequency of performance of simple tasks , which may reduced the specificity of this variable as an index of health literacy.et al., This variable contributed to support construct validity. The mean score for this prose task was low, with only around half of the participants scoring ⩾75%  to nine years (in 1986) and eventually 12 years (in 2009).SAHLPA-23 may be particularly useful to Portuguese health professionals and researchers as a quick method of obtaining an indication of health literacy in clinical and research settings, in order to anticipate potential problems with health literacy and facilitate more effective communication. Where possible socio-demographic data, such as years of education, should be collect and analysed together with this literacy measure.The SAHLPA-23 is an independent, effective and innovative self-administered tool for discriminating between adequate and inadequate health literacy. The SAHLPA-23 is a self-administered tool, unlike previous versions of the tool.


P < 0.01) that was driven by the loss of the small CR-ip interneurons (P < 0.01) in patients with SCH. The densities of the large CR-ip and of the NPY-ip interneurons were not significantly altered. The lower density detected could have been due to inflammation-induced degeneration. However, the state of microglial activation assessed by quantification of ionized calcium-binding adapter molecule 1 (Iba1)- and transmembrane protein 119 (TMEM119)-immunopositive cells showed no significant difference between patients with SCH and controls. Our results warrant further studies focussing on the role of CR-ip neurons and on the striatum being a possible hub for information selection and regulation of associative cortical fields whose function have been altered in SCH.Schizophrenia (SCH) and autism spectrum disorder (ASD) share several common aetiological and symptomatic features suggesting they may be included in a common spectrum. For example, recent results suggest that excitatory/inhibitory imbalance is relevant in the etiology of SCH and ASD. Numerous studies have investigated this imbalance in regions like the ventromedial and dorsolateral prefrontal cortex (DLPFC). However, relatively little is known about neuroanatomical changes that could reduce inhibition in subcortical structures, such as the caudate nucleus (CN), in neuropsychiatric disorders. We recently showed a significant decrease in calretinin-immunopositive (CR-ip) interneuronal density in the CN of patients with ASD without significant change in the density of neuropeptide Y-immunopositive (NPY-ip) neurons. These subtypes together constitute more than 50% of caudate interneurons and are likely necessary for maintaining excitatory/inhibitory balance. Consequently, and since SCH and ASD share characteristic features, here we tested the hypothesis, that the density of CR-ip neurons in the CN is decreased in patients with SCH. We used immunohistochemistry and qPCR for CR and NPY in six patients with schizophrenia and six control subjects. As expected, small, medium and large CR-ip interneurons were detected in the CN. We found a 38% decrease in the density of all CR-ip interneurons ( Schizophrenia (SCH) is a chronic and serious mental illness which puts an enormous burden on the individual, families, and society. Recent meta-analyses based on multinational surveys and robust longitudinal studies established the lifetime and 1-year prevalence of SCH at 0.4%  and 1.1%At the functional level, excitatory/inhibitory imbalance in the brain has been implicated in SCH , and mayIn our current study we primarily aimed to investigate the involvement of striatal interneurons in SCH. We focussed on the CN and two major interneuron populations, calretinin-immunopositive (CR-ip) and neuropeptide Y-immunopositive (NPY-ip) neurons which together constitute more than 50% of CN interneurons , and app1) and the Oxford Brain Bank (OBB). Eight cases with clinical diagnosis of SCH (based on DSM III and DSM IV) and six control subjects without history of psychiatric disorders were studied. All material was collected from donors from whom written informed consent had been obtained by the OBB or NBB for brain autopsy and use of material and clinical information for research purposes. The demographic characteristics of the cohort are shown in n = 12, p = 0.10, measured from the anterior commissure). Tissue blocks were also requested from the dorsolateral prefrontal cortex  and paraffin embedded tissue was provided from the medial frontal gyrus. Fresh-frozen tissue from the superior frontal gyrus was used for qPCR experiments.Tissue from subjects was provided by the Netherlands Brain Bank (NBB) Netherlands Institute for Neuroscience, Amsterdam  for 30 min. Antigen retrieval was applied by autoclaving the slides in citrate buffer  at 121°C for 10 min. The following primary antibodies were used: anti-calretinin , anti-neuropeptide Y , anti ionized calcium-binding adapter molecule 1  and anti-transmembrane protein 119  in Tris-buffered saline/Triton TM X-100 (pH 7.4) for 1 h (100 μl/section). Sections were then incubated with horseradish peroxidase-linked secondary antibody from the Envision Kit  for 1 h (100 μl/section) and labeling was visualized by DAB from the same Envision Kit applied for 90 s (100 μl/section). During incubation with primary and secondary antibodies slides were put into Sequenza System coverplates and rack . Two rinses with Tris-buffered saline/Triton TM X-100 (pH 7.4) were applied between the above-described steps of immunohistochemistry . Haematoxylin nuclear counterstain was applied for 20 s. Sections were dehydrated through a graded alcohol series and coverslipped with DePeX. No labeling was observed when primary antibodies were omitted from the protocol.Serial sections (6-μm thick) were cut in the coronal plane from paraffin-embedded blocks and mounted on slides. Consecutive sections were stained for the four antigens investigated. Our immunohistochemical analysis was done as described in detail in earlier studies , 2019. Bmsdlt-slide.dpag.ox.ac.uk). The regions of interest  were outlined using the ImageScope program  at 20× magnification and stored on a server  was dissected from individual samples and stored at −80°C. Total RNA was extracted with RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions, with genomic DNA removed by on-column DNase set (Qiagen). SuperScript III RT kit (Qiagen) was used for reverse transcription and the cDNA was then subjected to real-time quantitative PCR  with SYBR Green PCR Master Mix (Kapa Biosystems). Technical duplicates were included for all reactions. To select the internal references, seven candidate genes  were analyzed with NormFinder . The comt-test  was used to assess if the means of variables between the two diagnostic groups were significantly different. Fisher’s exact test was used to compare the distribution of gender between the groups. For correlation analyses Pearson’s correlation test was applied. Results of statistical comparisons presented are all based on unpaired, two-tailed Student’s t-tests if not stated otherwise.Results are presented as means ± standard error of the mean. One section per subject was used per immunostain . StatistIn order to control for demographic variables and to reveal which neuronal subtype was the most affected in the CN, the univariate analysis of variance [general linear model (GLM)] was applied. The densities of the three subtypes  and the total CR-ip population were tested separately. The densities of NPY-ip neurons, stained area fractions of Iba1 and TMEM119 and mRNA levels were also tested as dependent variables in separate univariate analyses of variance. Diagnosis was tested as a between-subjects factor and potential confounders  as covariates. Significance level was set to 5% (α = 0.05).N = 6). Three types of CR-ip neurons were found in the human CN in line with . We analyzed every caudate nucleus CR-ip neuron in our sections. Qualitative comparison of process numbers, branching and somal shape did not reveal conspicuous differences between the control and SCH groups , medium – 19.20 ± 0.14 μm , large – 31.27 ± 0.25 μm . The ratios of different CR-ip subtypes  were also very similar in both groups .We next compared the control CR-ip caudate neurons with 7,351 CR-ip neurons in patients with SCH  and subjects with SCH  that underlined the feasibility of the diameter measurements and compatibility of the two approaches.To evaluate the efficacy of measuring diameters instead of neuronal cross-sectional areas we investigated the correlation of these two variables by measuring 60 randomly selected neurons from each case . Similar to diameters, cross-sectional areas were not significantly different between the diagnostic groups: controls – 118.64 ± 3.12 μm2, controls: 1,944 ± 183 neurons/cm2, n = 12, p < 0.01, 2, controls: 262 ± 23 neurons/cm2, n = 12, p < 0.05, 2, controls: 172 ± 18 neurons/cm2, n = 12, p = 0.131, 2, controls: 2,378 ± 210 neurons/cm2, n = 12, p < 0.01, There was a 39% decrease in the density of small CR-ip neurons in subjects with SCH compared to controls  was applied. The three subtypes  and the total CR-ip population were tested separately. Possible confounders such as age, gender and post-mortem-interval were taken into consideration and their potentially significant effect on the CR-ip neuronal densities was also tested. This analysis returned no significant effects of the aforementioned variables on the densities of CR-ip neurons . On the n = 11) from a previously published dataset (n = 6). Because of the differences in terms of time of fixation in PFA and position of sampling between the cohorts, a GLM analysis was run with the following variables: age, gender, post-mortem interval, time of fixation and position of sampling. This analysis confirmed the significant effect of diagnosis on the density of the total CR-ip population  and particularly, on the density of the small CR-ip neurons . Cross-sectional areas of the CN were non-significantly smaller in subjects with SCH which if anything should have increased the density of CR-ip neurons. There was no statistically significant correlation between age and cross-sectional areas of the CN in the SCH group , in the CTR group , or even when these groups were merged . These data are shown as scatter plots in We compared the cross-sectional areas of the CN and found no significant difference between the diagnostic groups . We found a 6% higher density of NPY-ip neurons in patients with SCH, however, this was not statistically significant  in the GLM analysis from our previously published dataset  or in the TMEM119-ip stained area fraction , either. There was a significant positive correlation between Iba1-ip and TMEM119-ip stained area fractions measured in the CN .In order to determine if the decrease in CR-ip density was associated with increased inflammation we carried out immunohistochemical analysis of the microglial markers Iba1 and TMEM119. Qualitative assessments of Iba1-ip and TMEM119-ip microglia morphology by three independent observers did not reveal microglial morphology differences between controls and cases with SCH . MicroglThere were no significant effects of diagnosis or other variables  on the Iba1-ip and TMEM119-ip stained area fractions using GLM analysis . For the2, SCH: 2,264 ± 327.11 cells/cm2, n = 12, p = 0.985, 2, SCH: 119 ± 13.15 cells/cm2, n = 12, p = 0.582, We were interested whether the observed changes in interneuronal composition of the CN in SCH were mirrored by alterations in the region primarily connected to the pre-commissural head of the CN, the DLPFC. In order to detect the density of CR- and NPY-ip cortical neurons, immunohistochemistry was carried out on samples from the medial frontal gyrus, a subregion of the DLPFC. The dominant CR-ip neuronal type in this region had elongated and bipolar morphology  both in p = 0.061, Overall, we did not find any statistically significant changes regarding the transcript levels of CR and NPY in the DLPFC (Brodmann area 9) of patients with SCH . Howeverr = 0.103, n = 5; r = 0.086, n = 6, respectively, Spearmann’s rank correlation). These observations suggest that the decreased density of CR-ip neurons in SCH is independent of anti-psychotic treatment.We investigated if CR-ip neuronal densities in the CN or the DLPFC were correlated with medication history . There wIn this study, we demonstrated a strongly significant lower CR-ip CN interneuron density in SCH compared to controls, a major finding in the neuropathology of the condition. The importance of our result is underlined by the fact that no post-mortem neuronal subtype characterization has been carried out in the past 15 years concerning the CN in SCH. Nevertheless, our findings justify renewed interest in this effort.In support of our work, several independent studies using different techniques identified CR-ip interneurons in the CN as possibly important in SCH. CN interneurons have been implicated in SCH by high throughput sequencing data and meta-analysis of common variant genomic results . Anotherin vitro assays. Genome wide association studies identified a wide array of genetic mutations in synaptic scaffolding and ion-channel proteins in SCH  which, as mentioned above, was recently identified as a candidate locus in SCH . Deficits in SCH . Thus, aThere is a remarkable paucity of information on the electrophysiological properties, connectivity and function of CR-ip neurons in the CN. This may be because they are only 0.5% of neostriatal (caudate-putamen) neurons in rodents  and therPost-mortem neuropathological research on neuropsychiatric diseases is hindered by the scarcity of brain tissue available. Enthusiasm for conducting post-mortem studies in SCH is further decreased by the dogma that long-term neuroleptic treatment may obscure neuropathological findings. Specifically relevant to our study, enlarged basal ganglia previously found in SCH may be related to medication . An enlain vivo CN D2-receptor density between neuroleptic-naïve patients and patients receiving anti-psychotic treatment [median duration of treatment was 3.5 years  models  and did .5 years ]. At the.5 years . However.5 years  should b.5 years .2) than in theirs , Research Ethics Committee of the Hungarian Medical Research Council (45102-2/2016/EKU). The patients/participants provided their written informed consent to participate in this study.IA and FGS designed the study and wrote the manuscript. IA and BS conducted IHC and qPCR experiments. IA, VF, and TT contributed to quantification. DV and SC provided support in statistical analyses. IA prepared the figures and tables. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BA) of most eye drops is low since eye drops cannot be instilled beyond the capacity of the conjunctival sac due to its limited volume. Thus, traditional eye drops have low BA and a short duration of the drug on the ocular surface, so solutions to these problems are highly anticipated. In this study, we designed a sustained-release drug-delivery system (DDS) for TL nanoparticles. TL nanoparticles were prepared by bead mill treatment, and the gel formulations containing TL nanoparticles  were provided by carboxypolymethylene. The crystal structure of TL with and without bead mill treatment is the same, but the TL solubility in formulations containing nanoparticles was 5.3-fold higher compared with gel formulations containing TL microparticles (TL-MPs-Gel). The photo and thermal stabilities of TL-NPs-Gel are also higher than those of dissolved TL. Moreover, when TL-NPs-Gel is applied to the upper eyelid skin (outside), the TL is released as nanoparticles, and delivered to the lacrimal fluid through the meibomian glands. In addition, the TL release profile for TL-NPs-Gel was sustained over 180 min after the treatment. These findings can be used to develop a sustained-release DDS in the ophthalmic field.Eye drops containing Tranilast (TL), N- anthramilic acid, are used as an anti-allergic conjunctivitis drug in the ophthalmic field. Traditional eye drops are very patient compliant, although the bioavailability ( BA) for most eye drops is low, since the eye drops cannot be instilled beyond the capacity of the conjunctival sac due to its limited volume, and ocular functions, such as blinking, nasolacrimal drainage and tear turnover, also limit the BA of drugs instilled as eye drops [BA and a short duration on the ocular surface. Solutions to these problems are expected.Eye drops are considered to be the topical delivery route of drug administration for the treatment of ophthalmic diseases and are extensively utilized as pharmaceutical formulations . Althougye drops ,3. MoreoThe eyelid is the thin skin located on the ocular surface with the thickness on the human body <1 mm . There aThere are recent reports of systems to deliver drugs into skin tissue, such as in situ gels. Iontophoresis , nanostrTL, N- anthramilic acid, reduces extracellular matrix production, matrix metalloproteinase-1 secretion, transforming growth factor beta 1 (TGF-β1) secretion, cell migration, chemotaxis and collagen synthesis ,21,22. F2).Adult rabbits  were purchased from Shimizu Laboratory Supplies Co. Ltd.  The experimental protocol was approved by the Pharmacy Committee Guidelines for the Care and Use of Laboratory Animals in Kindai University . All experiments were performed following the guidelines for ARVO. In this study, the treatment of TL formulations was performed at 14:00, and 0.75% TL formulations (0.3 g) were applied to the shaved upper eyelid skin , and used as ophthalmic formulations containing TL nanoparticles (TL-NPs-Gel). In this study, formulations containing TL microparticles and dissolved TL (liquid TL) were also prepared and defined as TL-MPs-Gel and TL-liq-Gel , respectively. The TL-MPs-Gel and TL-liq-Gel were prepared as follows. TL-MPs-Gel: TL, MC and HPβCD were dispersed in distilled water and gelled with carboxypolymethylene. TL-liq-Gel: TL, MC and HPβCD were dissolved in 0.8% DMSO and gelled with carboxypolymethylene (Carbopol® 934). The ratios of TL, MC, and HPβCD in the TL formulations were 0.75%, 0.5% and 5%, respectively.Ophthalmic dispersions containing TL nanoparticles were prepared according to the previous reports ,32. TL pSamples were prepared as follows: mixtures of TL, MC and HPβCD were milled by the Bead Smash 12 as described above, and the milled TL was lyophilized, and used as samples. A powder X-ray diffraction (XRD) Mini Flex II  was used to analyze the crystal form, with the scanning rate, X-rays, and diffraction angles set to 10°/min, 30 kV and 15 mA, 5° to 90°, respectively.® ODS-3 column  at 35 °C, and detected at 230 nm. The mobile phase was 50 mM ammonium acetate and acetonitrile (80:20) at a flow rate of 0.25 mL/min.TL contents were measured by the HPLC method using an HPLC LC-20AT system . Ethyl p-hydroxybenzoate was used as an internal standard and the TL was added to 100 μL methanol containing ethyl p-hydroxybenzoate (3 mg/L). The mixture was injected into an InertsilThe TL formulations were diluted in distilled water and used as samples for measurement. The size dispersion of the TL-MPs-Gel was analyzed on a SALD-7100  with the refractive index set at 1.60-0.10i. In addition, the size distribution and number of nanoparticles in the TL-NPs-Gel were measured using a NANOSIGHT LM10  with the viscosity, wavelength and measurement time set to 1.27 mPa⋅s, 405 nm and 60 s, respectively. Atomic force microscopic (AFM) images were provided by a SPM-9700  according to previous report .TM MAX-XP Ultracentrifuge . The collected TL particles were dissolved in methanol, and the ratios of solid TL particles and dissolved TL were measured by the HPLC method described above.The solid TL particles and the dissolved TL in the TL formulations were separated by centrifuging at 100,000 g in an Optima2 florescent light (400–700 nm) at 25 °C for 24 h in the test for photostability. In the experiments for thermal stability, 0.3 g of TL formulations was kept at 60 °C for 10 weeks. The TL contents were measured by the HPLC method described above.The TL formulations (0.3 g) were stored under 58 W/mTL release from the TL formulations was evaluated as in previous studies using a Franz diffusion cell ,17,18,192 of shaved upper eyelids skin (outside), and samples of lacrimal fluid with or without meibum, or meibum only were collected. The Schirmer tear test strips were used to collect the lacrimal fluid in this study. Lacrimal fluid with meibum was collected from rabbits under the standard conditions by the insert the Schirmer tear test strips to the inside of eyelid. When lacrimal fluid without meibum was harvested, the outlet of the meibomian glands was covered with Schirmer tear test strips, a space was made between the eyelid and the ocular surface, and lacrimal fluid without meibum was collected by inserting the Schirmer tear test strips to inside of eyelid.The eyelids of the rabbits were shaved and 0.3 g of TL formulations (0.75%) were applied to the 1.7 cmP-values less than 0.05 considered significant. Differences were analyzed by ANOVA followed by the Student’s t-test and Dunnett’s multiple comparisons.Data are shown as the mean ± standard error (S.E.) with 2 florescent light, leaving a TL content of 38.9% after 24 h of irradiation. In addition, the thermal resistance for the dissolved TL in TL-liq-Gel was low, with the TL content decreased to 80.2% after 24 h at 60 °C. Both the photochemical and thermal resistances of the solid TL in TL-MPs-Gel and TL-NPs-Gel were attenuated in comparison with TL-liq-Gel. the viscosity of TL-MPs, TL-NPs and TL-liq-Gel were 14.4 ± 0.8 Pa∙s, 14.9 ± 0.9 Pa∙s, 11.3 ± 0.8 Pa∙s (n = 5), respectively.9 nanoparticles detected in the reservoir chamber. The residence time of traditional eye drops is short, and eye drops cannot be used when sleeping. Thus, the development of novel ophthalmic formulations to make sustained drug supplementation onto the ocular surface possible is highly expected. In this study, we designed a sustained-release ophthalmic formulation based on TL solid nanoparticles (TL-NPs-Gel) and investigated whether the application of TL-NPs-Gel to the eyelid can deliver TL into the lacrimal fluid.In the preparation of solid nanoparticles, previous reports have shown the importance of the selection of additives . We haveIn this study, we investigated whether the TL in TL-NPs-Gel is released as nanoparticles, and used the membrane with 220 nm pore size, because it is expected to see no micro- and nanoparticles released from TL formulations in the reservoir chamber. There are differences in the release rates for the three TL formulations A. A rate2, respectively  provides sustained TL supplementation to the lacrimal fluid, probably through the meibomian glands, to the ocular surface from the eyelid. These findings concerning a sustained-release ophthalmic formulation and the route of penetration through the meibomian glands provide significant information to develop ophthalmic formulations. Further studies are needed to develop a TL nano-delivery system through the eyelid, and it is important to clarify the mechanism of the trans-eyelid penetration of TL-NPs-Gel.


Peptides larger than 3–4 kDa, such as neuropeptide Y (NPY), orexin-B, and alpha-MSH, have practical issues that arise when conducting direct and sensitive quantitative liquid chromatography (LC) orbitrap-FT mass spectrometry (MS) due to their adsorption and low ionization efficiency, especially in standard solutions. A mixing solvent consisting of 0.5% trifluoroacetic acid (TFA) and 35–50% aq. acetonitrile was developed as the standard NPY for creating calibration curves, as well as a matrix to block the experimental tube surface to minimize adsorption. The mixture matrix effectively blocked non-specific adsorption of the standard peptides with tryptic digested bovine serum albumin (BSA)  and orexin-B (a large chain peptide). A sample containing 1 : 100 peptide:water was detected in the developed sample solution. Finally, 2 to 1,000 fmol/μL NPY could be analyzed quantitatively and reproducibly using conventional LC-MS. Parameters of the calibration curves, such as X-intercept, Bias (%), and relative standard deviation (RSD), were adjusted to optimize the sample solutions and the sensitive and quantitative LC-MS analyses. The present report describes the development of improved practical protocols for producing the calibration curves for sensitive quantitative liquid chromatography-mass spectrometry (LC-MS) analysis of neuropeptides.Neuropeptides are found in brain and central nervous system tissues and act as neurotransmitters and neurohormones. For example, neuropeptide Y (NPY), orexin-B, and α-MSH help regulate feeding behavior and control blood pressure.Calibration curves are important for sensitive quantitative LC-MS analyses. The experimental quality depends on the accuracy, precision, and detection limits of the calibration curve. In general, the amounts of biomolecules are so small that practical difficulties are encountered during quantitative analyses. Trace quantity analysis should be developed initially, followed by methods for experimental reproducibility and accuracy. If large amounts of bio-samples and target molecules are available, large-scale analyses can be performed under the exact and ideal calibration curves. Finally, quantitative LC-MS analyses of the biomolecules depend on the balance among sample availability, sensitivity, and reproducibility of the practical LC-MS system.The goal of the present study was to quantify small amounts of neuropeptides in brain tissue. These neuropeptides exist in very small quantities in brain tissue. The strategy for developing a sensitive quantitative LC-MS protocol involved optimization of the calibration curves parameters of X-intercept, Bias (%), and relative standard deviation. To reduce the LC-MS detection limits, X-intercept and Bias (%) values can be used to evaluate the experimental conditions. Relative standard deviation represents the reproducibility of the experiments. The LC-MS experiment parameters and preparation protocols were optimized for the sensitive quantitative LC-MS analysis of neuropeptides. Neuropeptides are expressed as precursor proteins that have no biological activity themselves. After the precursor proteins are cleaved and amidated enzymatically as post-translations, the bioactive peptides are generated. To study the maturation process of the neuropeptides, precursor peptides and their intermediates in each maturation step need to be detected and analyzed directly without artificial enzymatic digestion.Neuropeptide Y (NPY) is amidated at the C-terminal and contains 36 amino acids larger than 4 kDa. In sensitive LC-MS analysis of NPY and neuropeptides larger than 3 or 4 kDa, creating calibration curves is difficult due to the neuropeptide chemical properties. Large neuropeptides were speculated to form multimers or clusters easily in an aqueous standard solution, inducing non-specific adsorption to the experimental tube surface and reducing their ionization efficiency. Thus, the sensitive quantitative LC-MS analysis of NPY as a neuropeptide was optimized by focusing on the contents of the preparation solutions and matrix solutions.The NPY is soluble in water because NPY contains basic amino acids and functions as a secretory hormone. However, NPY exhibits carry-over in the LC-MS. A previous report described how to troubleshoot the NPY carry-over in an LC-MS system.Neuropeptide Y , trypsin-digested bovine serum albumin (BSA) MS standard (carboxymethyl-modified) , and HPLC grade acetonitrile  were used. MilliQ water  was used for all solutions. An aqueous solution containing 10.0 μM of NPY, orexin-B, and α-MSH was prepared as the stock solution. A series of 0.4, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 μM standard samples were prepared from the stock solution in the calibration curves of The experiments of entry 1–10, the trypsin-digested BSA was diluted with water at 10 fmol/μL concentration as a basic matrix solution. When the various matrix solutions were made in entry 1–10, the pipette tips were washed gently with the digested BSA basic matrix solution over five times. When the series of the standard samples were made in entry 1–10, the vials and pipette tips were washed gently with the corresponding matrix solutions to block the surface of the vials and pipette tips before starting the experiments. The series of the standard samples were diluted with serial dilution due to the possibility of the sample adsorption to the vials and pipette tips.A Nexera UPLC/HPLC system  was used in conjunction with a MS instrument. The sample-needle washing solution was 50% aq. acetonitrile in the auto-sampler. Aliquots of 1 μL of the standard and sample solutions were injected for the LC-MS experiments. An Aeris Peptide XB-C18  with a Security Guard Ultra guard column  was used at a flow rate of 0.3 mL/min. A YMC-Triart C8 metal-free column  was used at a flow rate of 0.2 mL/min. The gradient elution solutions used were 0.1% (v/v) formic acid/water (solution A) and 0.1% (v/v) formic acid/acetonitrile (solution B). The gradient programs are summarized in m/z 350–2,000 mass range, capillary temperature of 250°C, source heater temperature of 500°C, sheath gas flow rate of 50 (arbitrary unit specific to device), and AUX gas flow rate of 15 (arbitrary unit specific to device). The calibration curves were created using Xcalibur and QuanBrowser software of the FT-MS instrument . The peak area of the most abundant peak was used for their calibration curves. The target peak of NPY was at m/z=712.8547 ([M+6H]6+); that of orexin-B was at m/z=734.9128 ([M+4H]4+); that of α-MSH was at m/z=555.6055 ([M+3H]3+).All MS data were acquired on a Fourier transform (FT) orbitrap linear ion-trap hybrid (FT-Orbitrap Elite) MS instrument . The experimental parameters were: ion-source of ESI, positive-ion mode detection, MS resolution of 120,000 with Calibration curves are shown in 2) show the linearity of the calibration curves, and values of R and R2 greater than 0.998 are desirable for quantitative analysis, with values greater than 0.9998 for exact quantitative analysis. The R value for NPY was 0.99655 (Correlation coefficient (R) and coefficient of determination  shows the variation in the experimental data, and relative standard deviation (RSD) is the SD divided by the average as a percentage. The RSD at 0.5000 pmol-NPY was 2.19% , with goAlthough an ideal calibration curve passes through the origin, the experimental X-intercepts were 0.3000–0.4000 pmol for the calibration curves of NPY, orexin-B, and α-MSH (Bias (%) is the difference in the experimental average from the ideal values, calculated as follows: In general, Bias (%) values are about ±20% for quantitative analyses. The MS signals of the 5.0000-pmol samples were normalized as 100% of Bias, which caused the ideal calibration curve to pass through the origin. The Bias (%) values for the 2.0000-pmol samples of NPY, orexin-B, and α-MSH were approximately −20%; however, those of less concentrated samples were greatly reduced, especially NPY  values were used to optimize the sensitivity of the LC-MS analysis of NPY by reducing the adsorption of a target peptide. A practical issue involving the sample solutions of large chain peptides, such as NPY, is that their Bias (%) value is greatly reduced at lower concentrations of the calibration curves , which iWhen an aliquot of the sample absorbs non-specifically to the inside surface of the experimental tube, the adsorption ratio for highly concentrated samples is relatively low or negligible. That ratio in low concentration samples is relatively high, resulting in loss of the analytical sample. Therefore, the detection signals of LC-MS were much smaller in the lower concentration samples, causing an increase in the X-intercept and a significant reduction in Bias (%). Thus, sample solution preparation is very important for suppression of non-specific adsorption in sensitive quantitative LC-MS analysis. It has not been applied in reversed-phase liquid chromatography. Neuropeptide samples are applied to ODS or C-8 columns of LC systems, and so whole BSA cannot be used directly. Therefore, a tryptic-digested BSA fragment peptide mixture was used as the non-specific adsorption blocking matrix. All sample solutions in BSA is used as a blocking regent of non-specific adsorption of the analytes in biochemical experiments of proteins, such as immunohistochemistry and enzyme-linked immune sorbent assays (ELISA). In this study, these measures were applied while developing the LC system.The carry-over of peptides in the LC-MS system hindered sensitive quantitative LC-MS analysis, because the last sample carried over and was detected in the next experiments. The carry-over of NPY in LC-MS analysis has been resolved previously using washing programs in the auto-sampler and LC lines, changing LC columns, and not using a guard column.Aeris Peptide XB-C-18 was used for the peptide analyses  because the samples might not be attracted to the RP-LC column. The NPY was thought to have high hydrophobicity because of its high adsorption. Therefore, an attempt was made to dissolve NPY in 100% acetonitrile, but the attempt was not successful because the NPY aggregated. Since NPY may have amphiphilicity, 10–50% acetonitrile was added. The injection volume was fixed at 1 μL, which is so small that the sample can be held on a C-8 RP-LC column. Formic acid or TFA also was added to the solution because of the many basic amino acid residues of NPY. Adding acetonitrile and acid effectively improved the X-intercept value and Bias (%) in entry 2 (0.1% formic acid/10% aqueous acetonitrile solution). After the improvements, addition of 0.5% TFA and a few tens of percent acetonitrile was selected for preparing the contents of organic solvent and acids in entries 3 and 4.In experimental runs 1–5 at the lowest concentration , entry 1Adding acetonitrile and acid in the solutions shown as entries 2–4 improved the experimental reproducibility as shown in For the sample labeled as entry 4  at 0.0502 and RSD values also were good (entries 5 and 6, The matrix of entry 6  containeIn entry 7 , the matThus, entry 6  represenTrace quantity samples entries 8 and 9  were anaEnzymatic digested fragments of peptides and proteins usually are analyzed by LC-MS. These peptide fragment have greater ionization efficiency, no or much less adsorption, and no or much less self-aggregation compared to peptides larger than 3 or 4 kDa. Peptides larger than 4 kDa have lower ionization efficiency, and severe adsorption and self-aggregation issues in solution, which hinders reproducible and sensitive analyses. The experimental protocols developed here are essential for the analysis of large peptides. The sample preparation solution was an important factor in the sensitive quantitative LC-MS analysis of NPY. The sample solutions contained 0.5% TFA and some acetonitrile, and yet were analyzed by reversed-phase LC. The sample solutions also contained a non-specific adsorption blocking matrix with tryptic digested BSA fragment peptides and whole orexin-B. Two types of small and large peptides mixtures were very effective for stabilization of the solution sample and blockage of non-specific adsorption and self-aggregation. These results can be applied to improve analyses of other large peptides.


Levels were also lower in patients with poor outcomes compared to survivors , with an optimum cut-off 115 mg/L . Gc-globulin serum levels were variable according to ALF aetiology, i.e., lower in metabolic, infectious, or unknown causes compared to autoimmune and toxic causes. Gc-globulin serum levels were decreased in children with ALF and lower in those with poor outcomes compared with survivors. Gc-globulin serum levels were correlated with other known parameters used to evaluate the severity of ALF and could help to identify patients at high risk for poor outcomes.This study aimed to analyse vitamin D-binding protein (Gc-globulin) serum levels in acute liver failure (ALF) in children in relation to disease outcomes and correlations with other known markers used to evaluate the severity of ALF. Our study included 34 children (mean age 4.87 ± 5.30 years) with ALF of different causes  and 30 children without any liver injury (mean age 6.11 ± 4.26 years). The outcome was poor in 14 patients (41.18%), including one child with liver transplantation (2.94%). Serum Gc-globulin levels were significantly lower in ALF patients compared to the control group (151.57 ± 171.8 mg/L vs. 498.63 ± 252.50 mg/L;  Paediatric acute liver failure (ALF) is a severe condition defined according to the Paediatric Acute Liver Failure study group (PALFSG) criteria as a moderate coagulopathy  that cannot be corrected with parenteral vitamin K in the presence of encephalopathy, or severe coagulopathy INR > 2.0) in the absence of encephalopathy, in a previously healthy child .0 in the.Clinical and laboratory parameters are used to predict the evolution of ALF in children, such as encephalopathy, INR or coagulation factors V or VII ,5,6,7,8.Previous studies presented the role of new biological markers in predicting the evolution of ALF in adults. Gc-globulin serum levels were previously used to identify adults with high risk of fatal evolution, with different studies revealing that Gc-globulin levels were significantly lower in patients who died compared to those who survived. In ALF, Gc-globulin levels drop rapidly as the liver synthesises it, with some authors reporting concentrations that were 75% lower compared with adults without ALF ,17,18,19Our study aimed to assess serum levels of Gc-globulin in children with ALF and identify correlations with disease outcomes and other factors used to assess disease severity, namely, coagulation factors V and VII, INR, PELD score, and King’s College criteria.We enrolled 34 children hospitalised with ALF in this prospective study, who were followed-up in the 2nd Paediatric Clinic of the Emergency Clinical Hospital for Children, Cluj-Napoca, Romania, over a period of four years (January 2015–December 2018), alongside 30 children without liver illness (the control group). We used the PALFSG criteria to select the ALF patients included in this study, including those with jaundice, coagulopathy unable to be corrected by vitamin K infusion (INR > 1.5–1.9) and liver encephalopathy or INR > 2.0 without encephalopathy in a patient with no pre-existing liver condition ..p < 0.00p = 0.006763). Extreme Gc-globulin levels were observed in both the reference and surviving ALF patients. With the exclusion of these cases from our analysis, the comparisons were still significant regarding the Gc-globulin serum levels.The surviving ALF patients had a mean serum level below the normal lower limit (216.12 ± 199.69 mg/L), while those with fatal evolution exhibited Gc-globulin values way below the normal limit . Regardless of the cause of the ALF, the value of this parameter was lower overall in the ALF patients compared to the control group, where the average level was 498.63 ± 252.50 mg/L.INR levels, coagulation factors and Gc-globulin serum levels varied depending on the ALF aetiology. Gc-globulin serum levels were significantly lower in patients suffering from ALF due to metabolic disorder (42.54 ± 36.79 mg/L), infection (82.10 ± 14.11 mg/L) or unknown cause (87.89 ± 40.52 mg/L) compared to autoimmune (290.45 ± 200.40 mg/L) and toxic ALF (228.94 ± 234.02 mg/L)  and factor VII , and a weaker negative relationship with the PELD score . There was also a weak positive correlation with albumin level  and a negative association with INR .The correlation of Gc-globulin serum level with the other parameters already used in practice to assess the severity and prognosis of ALF was analysed, with Gc-globulin level showing a moderate–strong positive relationship with the levels of factor V (p < 0.000001), while the optimum cut-off was 163.5 mg/L with a sensitivity of 76.50%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 78.90%.To analyse the cut-off values of serum Gc-globulin in classifying ALF patients, including those with fatal evolution, we analysed area under curve (AUC) and receiver operating characteristic (ROC) curve values. p = 0.002329) and the optimum cut-off value for fatal evolution of ALF was 115 mg/L, with a sensitivity of 100%, a specificity of 50%, a positive predictive value of 58.30% and a negative predictive value of 100%.The analysis of the King’s College criteria in ALF children included in our study revealed that these criteria were positive in seven patients with fatal evolution (7/14) and in one who survived (1/20). Thus, the sensitivity of King’s College criteria was 50% and the specificity was 95% with a positive predictive value of 87.50% and a negative predictive value of 73.10%. The accuracy of King’s College criteria in our patients was 76.47%.Acute liver failure (ALF) in children is associated with a high mortality rate up to 50%) if a liver transplant is not urgently performed ,8,9. Var0% if a lGc-globulin  is a serum α2-globulin with a molecular weight of 52–59 kDa, which was described first by Hirschfeld in 1959 . Gc-globIn our study carried out on 34 children with ALF of various aetiologies, we assessed the serum levels of Gc-globulin in these children compared to the control group and correlated these levels with ALF outcomes and other severity-associated disease parameters. Gc-globulin serum levels were much lower in ALF patients compared to the control group, most likely a secondary effect of the aggression directed toward the liver, thereby affecting the hepatic synthesis processes. The average Gc-globulin serum level was also lower in patients with poor outcomes compared to those who survived .Only a few studies are available in the literature describing total Gc-globulin or actin-free Gc-globulin level reductions in cases of acute liver injury, and these works focused on adults ,38,39,40In our study, Gc-globulin levels in ALF varied with the aetiology. Gc-globulin levels were lower in ALF caused by metabolic, infectious, or unknown factors (possibly infection) compared to those caused by autoimmune and toxic factors, possibly due to greater severity of hepatic necrosis. In autoimmune ALF, even though the severity of the disease and the outcome could be poor, chronic evolution and the inflammatory process could explain the higher levels of Gc-globulin. This was also reported by studies performed in adults, where Gc-globulin levels were significantly lower in ALF patients and less so in those with chronic disease or chronic liver failure, such as autoimmune liver disease . Also, GWe also analysed the correlation between Gc-globulin and other parameters used for the prognosis of ALF and the PELD score. Low serum levels of Gc-globulin were highly significantly associated with low serum levels of factor V and factor VII and a high PELD score, which all indicate severe liver injury and a negative prognosis. No statistically significant correlation was revealed for albumin, which could be explained by the fact that Gc-globulin levels usually drop faster than albumin. This lack of correlation between the two related proteins, which are encoded by the same gene, was also revealed by Schiødt in patients who underwent liver transplantation.Our study showed an overall good performance of Gc-globulin in differentiating ALF in children (AUC 0.8921), with an optimum cut-off value of 163.5 mg/L. The performance of serum levels of Gc-globulin to differentiate children at risk for poor ALF outcomes was moderate (AUC 0.7642). The cut-off value for poor outcome without liver transplantation was 115 mg/L. Compared to the King’s College criteria, this cut-off value of serum Gc-globulin provides better sensitivity, but lower specificity and positive predictive value. When comparing this result to the literature, this cut-off was higher than in ALF in adults (80 mg/L) . AntoniaThe main limitation of our study was the small number of children with ALF included in the sample population, as this condition is rare in the paediatric population. Moreover, a larger number of ALF patients from different causes would increase the accuracy of the trend anaylsis of Gc-globulin in different etiologies. Still, this work constitutes one of only a few studies assessing Gc-globulin or other markers for the evaluation of the severity of ALF in children.In summary, our results confirmed in children the already existing data for adults regarding decreased serum levels of Gc-globulin in ALF and the correlations with the severity of liver injury. Gc-globulin serum levels could help to identify patients at high risk for poor outcomes. Additional studies are needed to further detail the prognosis potential of Gc-globulin serum levels in ALF in children.


The ACTS-CC 02 trial demonstrated that S-1 plus oxaliplatin (SOX) was not superior to tegafur-uracil and leucovorin (UFT/LV) in terms of disease-free survival (DFS) as adjuvant chemotherapy for high-risk stage III colon cancer . We now report the final overall survival (OS) and subgroup analysis according to the pathological stage (TNM 7th edition) for treatment efficacy.2 of UFT and 75 mg/day of LV on days 1-28, every 35 days, five cycles) or SOX . The primary endpoint was DFS and the patients’ data were updated in February 2020.Patients who underwent curative resection for pathologically confirmed high-risk stage III colon cancer were randomly assigned to receive either UFT/LV . In the subgroup analysis, the 5-year OS rates in patients with T4N2b disease were 51.0% and 64.1% in the UFT/LV and SOX groups, respectively .A total of 478 patients in the UFT/LV group and 477 patients in the SOX group were included in the final analysis. With a median follow-up time of 74.3 months, the 5-year DFS rate was 55.2% in the UFT/LV group and 58.1% in the SOX group [stratified hazard ratio (HR) 0.92; 95% confidence interval (CI) 0.76-1.11; Our final analysis reconfirmed that SOX as adjuvant chemotherapy is not superior to UFT/LV in terms of DFS in patients with high-risk stage III colon cancer. The 5-year OS rate was similar in the UFT/LV and SOX groups. •This final analysis reconfirmed that SOX is not superior to UFT/LV in high-risk stage III colon cancer in terms of DFS.•With a median follow-up time of 74.3 months, the 5-year OS rate was similar in both groups.•The 5-year OS rates in patients with T4N2b disease were 51.0% and 64.1% in the UFT/LV and SOX groups, respectively. N1 tumors with metastasis to the main lymph nodes have been associated with poorer outcomes than N2a tumors without metastasis to the main lymph nodes.Fluoropyrimidine plus oxaliplatin therapy is widely used as standard postoperative adjuvant chemotherapy for patients with stage III colon cancer worldwide. Among previous three phase III studies examining the superiority of oxaliplatin-based therapy, the NO16968 trial compared intravenous infusion of 5-fluorouracil (5-FU)/leucovorin (LV) with oral capecitabine and oxaliplatin (CAPOX), and the MOSAIC and NSABP C-07 trials compared 5-FU/LV with a combination therapy, including 5-FU/LV and oxaliplatin.The ACTS-CC trial showed that S-1 was noninferior to UFT/LV as adjuvant chemotherapy for stage III colon cancer.During the primary analysis with a median follow-up period of 58.4 months, SOX was not shown to be superior to UFT/LV in terms of disease-free survival (DFS) for primary endpoint in patients with high-risk stage III colon cancer , when there were only 187 events (19.6%) among 955 patients in terms of overall survival (OS).The ACTS-CC 02 trial is an open-label, multicenter, randomized phase III trial conducted in accordance with the ethical principles of the Declaration of Helsinki and was in compliance with the Japanese ethical guidelines for clinical studies. The study was approved by the Institutional Review Board of each participating institution. All patients provided written informed consent before enrollment. This trial is registered with the Japan Pharmaceutical Information Center (JapicCTI-101073) and Japan Registry of Clinical Trials (jRCTs031180351).Patients aged 20 to 80 years with curatively resected high-risk stage III  colon cancer were randomly assigned in a 1 : 1 ratio to the UFT/LV or SOX group, using the minimization method with the following stratification factors: tumor location (colon or upper rectum), the number of positive lymph nodes , and institution.2) and LV (75 mg/body) were orally administered daily in three divided doses for 28 consecutive days followed by a 7-day rest for a total of five cycles (25 weeks). In the SOX group, S-1 (80 mg/m2 daily) was orally administered in two divided doses, after dinner on day 1 to after breakfast on day 15 followed by a 7-day rest. Oxaliplatin (100 mg/m2) was infused intravenously on day 1 of each cycle. A total eight cycles (24 weeks) were administered. After completing the study treatment, patients were followed up according to a predefined surveillance schedule. The methods used in this trial have been described in detail previously.In the UFT/LV group, UFT , OS, and adverse events. DFS was defined as the period from the date of enrollment to the date of recurrence, secondary cancer, or death from any cause, whichever occurred first. Secondary cancer included metachronous cancers developing in not only the colorectum but also other organs.The 3-year DFS rate was estimated to be 65% for the UFT/LV group and 71.5% for the SOX group (HR 0.78). Five hundred and ten events in a total of 1186 enrolled patients were required to provide a statistical power of 80%  for detecting a difference between the groups after 3 years of follow-up. The primary analysis was conducted using the full analysis set on an intention-to-treat basis. We estimated time-dependent events using the Kaplan–Meier method. The stratified log-rank test using stratification factors excluding institution was used to compare the SOX with the UFT/LV group. All statistical analyses were performed using SAS version 9.13 or higher .https://doi.org/10.1016/j.esmoop.2021.100077). Demographic characteristics were well balanced between the groups . OS analysis was conducted on the basis of 254 deaths (26.2%) out of 955 patients, representing an increase of 78 deaths compared with the primary analysis. The causes of death were progressive disease in 217 patients, secondary cancer in 11 patients, and other reasons in 26 patients. The 5-year OS rate was 78.3% (95% CI 74.2-81.8) in the UFT/LV group and 79.1% (95% CI 75.1-82.5) in the SOX group. The stratified HR for OS was 0.97  or violated the Japanese ethical guidelines for clinical studies (two patients) were excluded. Finally, a total of 478 patients in the UFT/LV group and 477 patients in the SOX group were included in the updated analysis . However, a significant interaction was observed between the therapeutic effects and sex in the subgroup analysis of OS . The 5-year OS rates were also similar in the UFT/LV and SOX groups .As for DFS, the HR was lower in T4 disease than in T3 disease  and was even lower when with the N factors. As for OS, similar relationships between efficacy of SOX and T/N factors were observed; however, UFT/LV showed better OS than SOX in T3 disease. Although the results of subgroup analysis were not statistically significant due to the small number of patients, SOX had a favorable efficacy, in terms of DFS and OS, in patients with more advanced disease associated with factors such as T4N2b, and this might be a target population for SOX therapy. Further investigation in this population is required to elucidate the real efficacy of SOX in T4N2b population.The 5-year OS rate of stage IIIB and stage IIIC patients in the UFT/LV group of this study was comparable to that seen in the ACTS-CC trial that compared S-1 and UFT/LV in Japanese stage III colon cancer patients. The 5-year OS rate of stage IIIC patients in the SOX group was slightly higher than that of the patients in the ACTS-CC trial.In addition, circulating tumor DNA (ctDNA) testing for the detection of minimal residual disease targeting metastatic colorectal cancer has been effective for the detection of patient groups with a high recurrence risk. It was shown that the ctDNA-positive group has a significantly higher recurrence rate than the negative group, and the ctDNA positivity rate was high in cases with more advanced disease.2 was favorable compared with FOLFOX  in female patients, and the RDI was lower for female patients in both the SOX and FOLFOX groups.,2 with S-1 plus cisplatin as a first-line treatment for advanced gastric cancer, the RDI for SOX was lower in female patients, but there was no difference in the RDI of S-1 plus cisplatin according to patient sex, nor was there any difference in the OS on the basis of sex.The subgroup analysis of DFS in our study did not show any differences according to sex; however, the subgroup analysis of OS showed that SOX significantly improves the OS of female patients. Conversely, RDI was lower for female than for male patients. In the subgroup analysis of OS in the SOFT study that examined the first-line treatment of metastatic colorectal cancer, despite there being no significant interactions, SOX using oxaliplatin 130 mg/mOur current study had some limitations that should be considered while interpreting the findings. First, all patients enrolled in this study were Japanese and 90.6% of patients underwent D3 lymph node dissection; therefore, the results may not be generalizable to patients of other ethnicities. Second, 510 DFS events were required to provide a statistical power of 80% for detecting a difference between the groups. Because the target number of patients was not reached, 79 required events were not enough, ultimately leading to an underpowered study even after a median follow-up of >6 years. Finally, the ACTS-CC 02 trial compared different types of oral fluoropyrimidines, namely, S-1 and UFT/LV. It was not a study that tested the synergistic effect of adding oxaliplatin to fluoropyrimidine. At the time of planning the ACTS-CC 02 trial, the ACTS-CC trial was ongoing to demonstrate the noninferiority of S-1 to UFT/LV.This final analysis reconfirmed that SOX is not superior to UFT/LV in patients with high-risk stage III colon cancer in terms of DFS. The 5-year OS rate was similar in the UFT/LV and SOX groups. The oxaliplatin-based regimen could be more effective for DFS and OS in patients with advanced disease, such as T4N2b.